Nain Minu, Mukherjee Sriparna, Karmakar Sonali Porey, Paton Adrienne W, Paton James C, Abdin M Z, Basu Anirban, Kalia Manjula, Vrati Sudhanshu
Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.
Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi, India.
J Virol. 2017 Feb 28;91(6). doi: 10.1128/JVI.02274-16. Print 2017 Mar 15.
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target. Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.
日本脑炎病毒（JEV）是一种由蚊子传播的黄病毒，是东南亚病毒性脑炎的主要病因，并有成为全球病原体的可能性。在此，我们确定葡萄糖调节蛋白78（GRP78）是病毒进入和复制的一种重要宿主蛋白。利用来自小鼠神经元（Neuro2a）细胞的质膜组分，质谱分析确定GRP78是一种与重组JEV包膜蛋白结构域III相互作用的蛋白。发现GRP78在Neuro2a细胞、小鼠原代神经元和人上皮Huh-7细胞的质膜上表达。针对GRP78的抗体在所有三种细胞类型中均显著抑制JEV进入，表明该蛋白在病毒进入中起重要作用。通过小干扰RNA（siRNA）耗竭GRP78显著阻断JEV进入Neuro2a细胞，进一步支持其在病毒摄取中的作用。免疫荧光研究显示在病毒感染的细胞中GRP78与JEV包膜蛋白广泛共定位。这种相互作用也通过免疫沉淀研究得到证实。此外，GRP78在JEV复制中显示出重要作用，因为用特异性切割GRP78的枯草杆菌蛋白酶细胞毒素在病毒进入后处理细胞导致病毒RNA复制和蛋白质合成大幅减少，从而使细胞外病毒滴度显著降低。我们的结果表明，GRP78作为HSP70家族的一种内质网伴侣蛋白，是参与JEV生命周期多个步骤的一种新型宿主因子，可能是一个潜在的治疗靶点。近年来，由黄病毒引起的蚊媒疾病迅速传播。黄病毒科包括西尼罗河病毒、登革热病毒、日本脑炎病毒和寨卡病毒，它们是对公共卫生的主要威胁，并有成为全球病原体的可能性。JEV是东南亚几个地区病毒性脑炎的主要病因，主要影响儿童人群，死亡率很高。本研究的重点是确定关键的宿主因子，针对这些因子可削弱病毒感染并最终导致开发有效的抗病毒药物。我们已经确定了一种细胞蛋白GRP78，它在病毒进入和病毒复制这两个病毒生命周期的关键步骤中发挥双重作用，因此是一种新型宿主因子，可能是一个潜在的治疗靶点。
